Design of a Receptor Labeling Angiotensin II Analogue Containing a 3'hydroxy-Tyrosine in Position 4

摘要

Biologically active G Protein Coupled Receptor (GPCR) structures are not yet accessible to analytical methods. The human angiotensin II type 1 receptor (hAT1) has been investigated with photoaffinity labeling approaches using Angiotensin II (DRVYIHPF, Angll) analogues with biologically acceptable photolabile moieties. The radio iodinated photolabile ligand would bind with high affinity to the hAT1 receptor and UV irradiation would covalently link the ligand to the receptor. Following chemical and enzymatic digestions direct ligand receptor interactions could be elucidated. Many ligand-receptor contact points were found for most ligand residues; nevertheless, the central amino acid of Angll, the Tyrosine 4 residue could never be addressed due to inadequate SAR results with all attempted photolabile substitutions. For labeling experiments to succeed we needed an analogue with high specific affinity to its cognate receptor, a labeling moiety inserted in the analogue and a revealing moiety to identify the labeled receptor. To remediate this difficulty a DOPA (3', 4'dihydroxyphenylalanine or 3'-hydroxy-tyrosine) was introduced into position 4 of Angll, maintaining low nM receptor affinity (Table 1). Once oxidized with NaIO4, the DOPA moiety may form a covalent bond to a Cysteine residue of the target receptor trough a Michael addition of the free thiol group (Figure 1). The oxidized DOPA forms an electrophilic ortho-quinone that reacts with nearby nucleophiles such as Cysteine or Histidine and through a Schiff base formation with Lysine [1,2]. This analogue can thus maintain native like binding and permit the oxidative labeling of hAT1.